PROJECT SUMMARY Cells have evolved intricate enzymatic machineries that help them exist and survive redox stresses in their microenvironment. Enzymatic redox sense, signal, and response mechanisms are critical for a diverse set of physiological processes in all forms of life ranging from bacteria and plants to humans. Unlike other cellular signaling processes, redox signaling involves highly reactive reagents such as nitric oxide (NO), carbon monoxide (CO), and reactive oxygen species that raise concerns regarding the potential of these reagents for participation in other non-specific reactions. Yet, such side reactions are uncommon under physiological conditions suggesting high specificity and selectivity of enzymatic redox signal transduction pathways. In turn, we ask the following two pertinent questions: a) What makes redox signaling pathways so specific? and b) Can we rationally and systematically rewire redox signal transduction pathways to re-instate/disrupt cellular redox balance? These questions have been largely overlooked from the chemical biology and bioinorganic chemistry perspective, despite the fact that redox imbalances are responsible for a variety of diseases ranging from neurological disorders to cancer. Our lab focuses on these paradigm shifting questions and the long-term goal of our research program is to develop molecular strategies that rewire sensing/signaling mechanisms of biological redox reagents involved in human health and disease. In this proposal, we focus on DosS-DosR enzymatic signaling pathway in mycobacteria that senses NO/CO in its microenvironment and signals cellular transition into a non-replicating, dormant state. The DosS-DosR system has three components ? a heme iron sensing domain that binds to NO/CO, a zinc-dependent signaling kinase domain that communicates the binding event and a response domain that binds to DNA and turns on dormancy. Using our combined expertise in metalloprotein structure-function, protein engineering, enzymology and spectroscopy, we will devise mutagenic, metal-substitution and peptidomimetic-based approaches to rewire DosS-DosR sense, signal and response domains. Proof-of-concept studies conducted in our laboratory have demonstrated successful rewiring of DosS- DosR redox sensing function via structure-guided rational protein design. Future work will unravel the molecular mechanisms of redox rewiring and its implications on cellular physiology and phenotypic responses. Our findings, will not only provide a fundamental understanding of cellular redox sense/signal/response mechanisms, but also inform future methodologies for treatment and prevention of redox-related diseases. Health Relevance: Maintenance of a normal intracellular redox status is crucial for regulating physiological responses. Any imbalance in this status results in a variety of acute and chronic degenerative diseases such as cancer, cardiovascular and neurological disorders. Our research program aims to design molecular approaches that rewires the ability of cells to sense and signal redox changes in their environment. These approaches could be applied to re-instate cellular redox homeostasis in diseased states.